Nucleotide sequences coding for the Dep33 efflux protein

ABSTRACT

The invention provides nucleotide sequences from Coryneform bacteria coding for the dep33 efflux protein and a process for the fermentative preparation of amino acids using bacteria in which the dep33 efflux protein is attenuated.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to German Application No.DE 10044707.4, which was filed on Sep. 9, 2000 and German ApplicationNo. DE 10112430.9, which was filed on Mar. 15, 2001; the entire contentsof both documents are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention provides nucleotide sequences from Coryneformbacteria coding for the Dep33 protein and a process for the fermentativepreparation of amino acids using bacteria in which the dep33 effluxprotein is attenuated.

[0004] 2. Discussion of the Background

[0005] L-amino acids, in particular L-lysine, are used in human medicineand in the pharmaceutical industry, in the foodstuffs industry and veryparticularly in animal nutrition.

[0006] It is known that amino acids can be prepared by the fermentationof strains of coryneform bacteria, in particular Corynebacteriumglutamicum. Due to the importance of this area, constant efforts aremade to improve the method of preparation. Process improvements mayrelate to fermentation technology measures such as, for example,stirring and supplying with oxygen, or the composition of the nutrientmedia such as, for example, the sugar concentration during fermentation,or working up to the product form by, for example, ion exchangechromatography, or the intrinsic performance properties of themicroorganism itself.

[0007] Methods of mutagenesis and selection are used to improve theoutput properties of these microorganisms. Strains that are resistant toantimetabolites or are auxotrophic for regulatory significantmetabolites and which produce amino acids can be obtained with thesemethods.

[0008] Methods of recombinant DNA engineering have also been used forimproving Corynebacterium strains ability to produce L-amino acid byamplifying individual amino acid biosynthesis genes and examining theeffects on amino acid production.

[0009] However, there remains a critical need for improved methods ofproducing L-amino acids and thus for the provision of strains ofbacteria producing higher amounts of L-amino acids. On a commercial orindustrial scale even small improvements in the yield of L-amino acids,or the efficiency of their production, are economically significant.Prior to the present invention, it was not recognized that attenuationof the dep33 gene encoding the efflux protein Dep33 would improveL-amino acid yields.

SUMMARY OF THE INVENTION

[0010] An object of the present invention is to provide novel measuresfor the improved production of L-amino acids or amino acid, where theseamino acids include L-asparagine, L-threonine, L-serine, L-glutamate,L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine,L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine,L-tryptophan, L-arginine and the salts (monohydrochloride or sulfate)thereof.

[0011] One object of the present invention is providing a novel processfor improving the fermentative production of said L-amino acids,particularly L-lysine. Such a process includes enhanced bacteria,preferably enhanced Coryneform bacteria, which express attenuatedamounts Dep33 efflux protein or protein that has Dep33 efflux activity.

[0012] Thus, another object of the present invention is providing such abacterium, which expresses an attenuated amount of Dep33 efflux proteinor gene products of the dep33 gene.

[0013] Another object of the present invention is providing a bacterium,preferably a Coryneform bacterium, which expresses a polypeptide thathas an attenuated Dep33 efflux protein activity.

[0014] Another object of the invention is to provide a nucleotidesequence encoding a polypeptide having the Dep33 efflux proteinsequence. One embodiment of such a sequence is the nucleotide sequenceof SEQ ID NO: 1.

[0015] A further object of the invention is a method of making Dep33efflux protein or an isolated polypeptide having a Dep33 efflux proteinactivity, as well as use of such isolated polypeptides in the productionof amino acids. One embodiment of such a polypeptide is the polypeptidehaving the amino acid sequence of SEQ ID NO: 2.

[0016] Other objects of the invention include methods of detectingnucleic acid sequences homologous to SEQ ID NO: 1, particularly nucleicacid sequences encoding polypeptides that have Dep33 efflux proteinactivity, and methods of making nucleic acids encoding suchpolypeptides.

[0017] The above objects highlight certain aspects of the invention.Additional objects, aspects and embodiments of the invention are foundin the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWING

[0018]FIG. 1: Map of the plasmid pCR2.dep33int.

DETAILED DESCRIPTION OF THE INVENTION

[0019] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art of molecular biology. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described herein. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In addition, the materials, methods, andexamples are illustrative only and are not intended to be limiting.

[0020] Reference is made to standard textbooks of molecular biology thatcontain definitions and methods and means for carrying out basictechniques, encompassed by the present invention. See, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1989), Current Protocols in MolecularBiology, Ausebel et al (eds), John Wiley and Sons, Inc. New York (2000)and the various references cited therein.

[0021] “L-amino acids” or “amino acids” as used herein mean one or moreamino acids, including their salts, chosen from the group L-asparagine,L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine,L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine,L-phenylalanine, L-histidine, L-lysine, L-tryptophane and L-arginine.L-lysine is particularly preferred.

[0022] When L-lysine or lysine is mentioned in the following, this isintended to mean not only the bases but also salts such as e.g. lysinemonohydrochloride or lysine sulfate.

[0023] The invention provides a polynucleotide isolated from coryneformbacteria and containing a polynucleotide sequence coding for the dep33gene, chosen from the group consisting of

[0024] a) a polynucleotide which is at least 70% identical to apolynucleotide which codes for a polypeptide which contains the aminoacid sequence in SEQ ID No. 2,

[0025] b) a polynucleotide which codes for a polypeptide which containsan amino acid sequence which is at least 70% identical to the amino acidsequence in SEQ ID No. 2,

[0026] c) a polynucleotide which is complementary to the polynucleotidesin a) or b), and

[0027] d) a polynucleotide containing a sequence of at least 15consecutive nucleotides from the polynucleotide sequence in a), b) orc),

[0028] wherein the polypeptide preferably has the activity of the effluxprotein Dep33.

[0029] The invention also provides the polynucleotide mentioned above,wherein it is preferably a replicable DNA containing:

[0030] (i) the nucleotide sequence given in SEQ ID No.1, or

[0031] (ii) at least one sequence which corresponds to sequence (i)within the range of degeneracy of the genetic code, or

[0032] (iii) at least one sequence which hybridizes with sequences whichare complementary to sequences (i) or (ii), and optionally

[0033] (iv) functionally neutral sense mutations in (i).

[0034] The invention also provides:

[0035] a replicable polynucleotide, in particular DNA, containing thenucleotide sequence shown in SEQ ID No.1;

[0036] a polynucleotide which codes for a polypeptide which contains theamino acid sequence shown in SEQ ID No. 2;

[0037] a vector containing part of the polynucleotide according to theinvention, but at least 15 consecutive nucleotides from the claimedsequence,

[0038] and coryneform bacteria in which the dep33 gene is attenuated, inparticular by an insertion or a deletion.

[0039] The invention also provides polynucleotides which consistsubstantially of a polynucleotide sequence which are obtainable by thescreening, by means of hybridization, of a suitable gene library from acoryneform bacterium which contains the complete gene or a part thereof,with a probe which contains the sequence in the polynucleotide accordingto the invention in accordance with SEQ ID No.1 or a fragment thereofand isolating the polynucleotide sequence mentioned.

[0040] Polynucleotides which contain sequences in accordance with theinvention are suitable as hybridization probes for RNA, cDNA and DNA, inorder to isolate nucleic acids or polynucleotides or genes of fulllength which code for the efflux protein Dep33, or in order to isolatenucleic acids or genes which exhibit a high similarity to the sequencein the dep33 gene.

[0041] Furthermore, polynucleotides which contain the sequences inaccordance with the invention are also suitable as primers, with the aidof which, and using the polymerase chain reaction (PCR), the DNA ofgenes which code for the efflux protein Dep33 can be prepared.

[0042] Those oligonucleotides which are used as probes or primerscontain at least 25, 26, 27, 28, 29 or 30, preferably at least 20, 21,22, 23 or 24, very particularly preferably at least 15, 16, 17, 18 or 19consecutive nucleotides. Oligonucleotides with a length of at least 31,32, 33, 34, 35, 36, 37, 38, 39 or 40, or at least 41, 42, 43, 44, 45,46, 47, 48, 49 or 50 nucleotides are also suitable. Optionally,oligonucleotides with a length of at least 100, 150, 200, 250 or 300nucleotides are also suitable.

[0043] “Isolated” means separated from its natural surroundings.

[0044] A “polynucleotide” generally refers to polyribonucleotides andpolydeoxyribonucleotides, wherein these may be non-modified RNA or DNAor modified RNA or DNA.

[0045] Polynucleotides according to the invention include apolynucleotide in accordance with SEQ ID No. 1 or a fragment preparedtherefrom and also those which are at least 70% to 80%, preferably atleast 81% to 85%, particularly preferably at least 86% to 90%, and veryparticularly preferably at least 91%, 93%, 95%, 97% or 99% identical tothe polynucleotide in accordance with SEQ ID No. 1 or a fragmentprepared therefrom.

[0046] “Polypeptides” are understood to be peptides or proteins whichcontain two or more amino acids linked via peptide bonds.

[0047] Polypeptides according to the invention include a polypeptide inaccordance with SEQ ID No. 2, in particular those with the biologicalactivity of the efflux protein Dep33 and also those which are at least70% to 80%, preferably at least 81% to 85%, particularly preferably atleast 86% to 90%, and very particularly preferably at least 91%, 93%,95%, 97% or 99% identical to the polypeptide in accordance with SEQ IDNo. 2 and have the activity mentioned above.

[0048] Furthermore, the invention provides a process for thefermentative preparation of amino acids chosen from the groupL-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine,L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine,L-phenylalanine, L-histidine, L-lysine, L-tryptophane and L-arginine,using coryneform bacteria, in particular those which already produceamino acids and in which the nucleotide sequences coding for the dep33gene are attenuated, in particular switched off or expressed at a lowlevel.

[0049] In this context, the expression “attenuation” describes thereduction or switching off of the intracellular activity of one or moreenzymes (proteins) in a microorganism which are coded by thecorresponding DNA, for example by using a weak promoter or by using agene or allele which codes for a corresponding enzyme with a lowactivity or inactivates the corresponding gene or enzyme (protein) andoptionally combining these measures.

[0050] As a result of the attenuation measures, the activity orconcentration of the corresponding protein is generally lowered to 0 to75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity orconcentration of the wild type protein, or of the activity orconcentration of the protein in the initially used microorganism.

[0051] Microorganisms which are provided by the present invention canproduce amino acids from glucose, saccharose, lactose, fructose,maltose, molasses, starch, cellulose or from glycerin and ethanol. Theyare representatives of coryneform bacteria, in particular of the genusCorynebacterium. From among the genus Corynebacterium, the speciesCorynebacterium glutamicum has to be mentioned in particular, this beingrecognized by a person skilled in the art for its ability to produceL-amino acids.

[0052] Suitable strains of the genus Corynebacterium, in particular ofthe species Corynebacterium glutamicum (C. glutamicum), are inparticular the known wild type strains

[0053]Corynebacterium glutamicum ATCC13032

[0054]Corynebacterium acetoglutamicum ATCC15806

[0055]Corynebacterium acetoacidophilum ATCC13870

[0056]Corynebacterium melassecola ATCC17965

[0057]Corynebacterium thermoaminogenes FERM BP-1539

[0058]Brevibacterium flavum ATCC14067

[0059]Brevibacterium lactofermentum ATCC13869 and

[0060]Brevibacterium divaricatum ATCC14020

[0061] and L-amino acid-producing mutants or strains prepared therefrom.

[0062] Preferably, a bacterial strain with attenuated expression of adep33 gene that encodes a polypeptide with Dep33 efflux protein activitywill improve amino acid yield at least 1%.

[0063] The new dep33 gene coding for the efflux protein Dep33 wasisolated from C. glutamicum.

[0064] In order to isolate the dep33 gene, or also other genes, from C.glutamicum, a gene library from this microorganism is first compiled inEscherichia coli (E. coli). The compilation of gene libraries isdescribed in generally known textbooks and manuals. The text book byWinnacker: Gene und Klone, Eine Einführung in die Gentechnologie (VerlagChemie, Weinheim, Germany, 1990), or the manual by Sambrook et al.:Molecular Cloning, A Laboratory Manual (Cold Spring Harbor LaboratoryPress, 1989) may be mentioned as examples. A very well-known genelibrary is that of the E. coli K-12 strain W3110, which was compiled byKohara et al. (Cell 50, 495-508 (1987)) in λ-vectors. Bathe et al.(Molecular and General Genetics, 252:255-265, 1996) describe a genelibrary from C. glutamicum ATCC13032, which was compiled with the aid ofthe cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of theNational Academy of Sciences USA, 84:2160-2164) in E. coli K-12 strainNM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575).

[0065] Again, Börmann et al. (Molecular Microbiology 6(3), 317-326(1992)) describe a gene library from C. glutamicum ATCC13032 obtainedusing the cosmid pHC79 (Hohn and Collins, 1980, Gene 11, 291-298).

[0066] To prepare a gene library from C. glutamicum in E. coli, plasmidssuch as pBR322 (Bolivar, 1979, Life Sciences, 25, 807-818) or pUC9(Vieira et al., 1982, Gene, 19:259-268) may also be used. Particularlysuitable hosts are those E. coli strains which are restriction andrecombination defective such as, for example, the strain DH5αmcr whichwas described by Grant et al. (Proceedings of the National Academy ofSciences USA, 87 (1990) 4645-4649). The long DNA fragments cloned withthe aid of cosmids or other vectors are then again subcloned in suitablevectors commonly used for DNA sequencing and then sequenced, as isdescribed e.g. in Sanger et al. (Proceedings of the National Academy ofSciences of the United States of America, 74:5463-5467, 1977).

[0067] The DNA sequences obtained may then be examined using knownalgorithms or sequence analysis programs such as e.g. the one fromStaden (Nucleic Acids Research 14, 217-232(1986)), the one from Marck(Nucleic Acids Research 16, 1829-1836 (1988)) or the GCG program fromButler (Methods of Biochemical Analysis 39, 74-97 (1998)).

[0068] Additionally, methods employing DNA chips, microarrays or similarrecombinant DNA technology that enables high throughput screening of DNAand polynucleotides which encode the Dep33 efflux protein orpolynucleotides with homology to the dep33 gene as described herein.Such methods are known in the art and are described, for example, inCurrent Protocols in Molecular Biology, Ausebel et al (eds), John Wileyand Sons, Inc. New York (2000).

[0069] The new DNA sequence from C. glutamicum, coding for the dep33gene, was found and, as SEQ ID No. 1, is a constituent of the presentinvention. Furthermore, the amino acid sequence for the correspondingprotein was derived from the available DNA sequence using the methodsdescribed above. SEQ ID No. 2 gives the amino acid sequence in the dep33gene product which is obtained.

[0070] Coding DNA sequences which are produced from SEQ ID No. 1 by thedegeneracy of the genetic code are also a constituent of the presentinvention. In the same way, DNA sequences which hybridise with SEQ IDNo. 1 or parts of SEQ ID No. 1, are a constituent of the invention.Furthermore, in the specialist field, conservative amino acidreplacements, such as e.g. replacing glycine by alanine or aspartic acidby glutamic acid, in proteins are known as sense mutations which do notlead to any fundamental change in the activity of the protein, i.e. theyare functionally neutral. Furthermore, it is known that changes at theN-terminal and/or C-terminal of a protein does not substantially impairits function and may even stabilise it. A person skilled in the art mayfind information about this, inter alia, in Ben-Bassat et al. (Journalof Bacteriology 169:751-757 (1987)), in O'Regan et al. (Gene 77:237-251(1989)), in Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)), inHochuli et al. (Bio/Technology 6:1321-1325 (1988)) and in well-knowntextbooks on genetics and molecular biology. Amino acid sequences whichare produced from SEQ ID No. 2 in an appropriate manner are also aconstituent of the invention.

[0071] In the same way, DNA sequences which hybridize with SEQ ID No. 1or parts of SEQ ID No. 1 are a constituent of the invention. Finally,DNA sequences which are produced from SEQ ID No. 1 by the polymerasechain reaction (PCR) using primers are a constituent of the invention.These types of oligonucleotides typically have a length of at least 15nucleotides.

[0072] Instructions for identifying DNA sequences by means ofhybridization can be found by a person skilled in the art, inter alia,in the manual “The DIG System Users Guide for Filter Hybridization” fromBoehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al.(International Journal of Systematic Bacteriology 41: 255-260 (1991)).Hybridization takes place under stringent conditions, which means thatthe only hybrids formed are those in which the probe and targetsequence, i.e. the polynucleotides treated with the probes, are at least70% identical. It is known that the stringency of hybridization,including the washing step, is affected or determined by varying thebuffer composition, the temperature and the salt concentration. Thehybridization reaction is preferably performed at relatively lowstringency as compared with the washing step (Hybaid HybridisationGuide, Hybaid Limited, Teddington, UK, 1996).

[0073] For the hybridization reaction, for example, a 5×SSC-buffer maybe used at a temperature of about 50° C.-68° C. Probes may then alsohybridize with polynucleotides which are less than 70% identical to thesequence in the probe. These hybrids are less stable and are removed bywashing under stringent conditions. This may be achieved, for example,by lowering the salt concentration to 2×SSC and optionally then to0.5×SSC (The DIG System User's Guide for Filter Hybridisation,Boehringer Mannheim, Mannheim, Germany, 1995), wherein a temperature ofabout 50° C.-68° C. is used. It is also optionally possible to lower thesalt concentration to 0.1×SSC. By a stepwise increase in thehybridization temperature from 50° C. to 68° C., in steps of about 1 -2°C., polynucleotide fragments can be isolated which are, for example, atleast 70% or at least 80% or at least 90% to 95% identical to thesequence in the probe used. Further instructions for hybridization, inthe form of so-called kits, are commercially available (e.g. DIG EasyHyb from Roche Diagnostics GmbH, Mannheim, Germany, Catalogue No.1603558).

[0074] A person skilled in the art may find instructions for theamplification of DNA sequences using the polymerase chain reaction(PCR), inter alia, in the manual by Gait: Oligonucleotide Synthesis: APractical Approach (IRL Press, Oxford, UK, 1984) and in Newton andGraham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).

[0075] It was found that Coryneform bacteria produce amino acids in animproved manner following attenuation of the dep33 gene.

[0076] To produce attenuation, either expression of the dep33 gene orthe catalytic properties of the enzyme protein may be reduced orswitched off. Optionally, both measures may be combined.

[0077] A reduction in gene expression may take place by appropriateculture management or by genetic modification (mutation) of the signalstructures for gene expression. Signal structures for gene expressionare, for example, repressor genes, activator genes, operators,promoters, attenuators, ribosome bonding sites, the start codon andterminators. A person skilled in the art may find information aboutthese e.g. in patent application WO 96/15246, in Boyd and Murphy(Journal of Bacteriology 170: 5949 (1988)), in Voskuil and Chambliss(Nucleic Acids Research 26: 3548 (1998), in Jensen and Hammer(Biotechnology and Bioengineering 58: 191 (1998)), in Patek et al.(Microbiology 142: 1297 (1996)), Vasicova et al. (Journal ofBacteriology 181: 6188 (1999)) and in well-known textbooks on geneticsand molecular biology such as e.g. the textbook by Knippers (“MolekulareGenetik”, 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) orthe book by Winnacker (“Gene und Klone”, VCH Verlagsgesellschaft,Weinheim, Germany, 1990).

[0078] Mutations which lead to a change or reduction in the catalyticproperties of enzyme proteins are known from the prior art; the papersby Qiu and Goodman (Journal of Biological Chemistry 272: 8611-8617(1997)), Sugimoto et al. (Bioscience Biotechnology and Biochemistry 61:1760-1762 (1997)) and Möckel (“Die Threonindehydratase ausCorynebacterium glutamicum: Aufhebung der allosterischen Regulation undStruktur des Enzyms”, Jülich Research Center, Report, Jü-2906,ISSN09442952, Jülich, Germany, 1994) may be mentioned as examples.Reviews of the subject can be found in well-known textbooks on geneticsand molecular biology such as e.g. the book by Hagemann (“AllgemeineGenetik”, Gustav Fischer Verlag, Stuttgart, 1986).

[0079] Suitable mutations are transitions, transversions, insertions anddeletions. Depending on the effect of amino acid replacement on theenzyme activity, reference is made to missense mutations or nonsensemutations. Insertions or deletions of at least one base pair (bp) in agene lead to frame shift mutations, as a result of which incorrect aminoacids are incorporated or translation is terminated prematurely.Deletions of several codons lead typically to complete failure of enzymeactivity. Instructions for producing these types of mutations are partof the prior art and can be found in well-known textbooks on geneticsand molecular biology such as e.g. the textbook by Knippers (“MolekulareGenetik”, 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995),the book by Winnacker (“Gene und Klone”, VCH Verlagsgesellschaft,Weinheim, Germany, 1990) or the book by Hagemann (“Allgemeine Genetik”,Gustav Fischer Verlag, Stuttgart, 1986).

[0080] A common method of mutating genes in C. glutamicum is the methodof gene disruption and gene replacement described by Schwarzer andPühler (Bio/Technology 9, 84-87 (1991)).

[0081] With the method of gene disruption a central part of the codingregion of the gene being considered is cloned in a plasmid vector whichcan replicate in a host (typically E. coli), but not in C. glutamicum.Suitable vectors are, for example, pSUP301 (Simon et al., Bio/Technology1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73(1994)), pK18mobsacB or pK19mobsacB (Jäger et al., Journal ofBacteriology 174: 5462-65 (1992)), pGEM-T (Promega corporation, Madison,Wis., USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry269:32678-84; U.S. Pat. No. 5,487,993), pCR® Blunt (Invitrogen,Groningen, Netherlands; Bernard et al., Journal of Molecular Biology,234: 534-541 (1993)) or pEM1 (Schrumpf et al, 1991, Journal ofBacteriology 173:4510-4516). The plasmid vector which contains thecentral part of the coding region of the gene is then transferred byconjugation or transformation into the desired strain of C. glutamicum.The method of conjugation is described, for example, in Schäfer et al.(Applied and Environmental Microbiology 60, 756-759 (1994)). Methods fortransforming are described, for example, in Thierbach et al. (AppliedMicrobiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan(Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMSMicrobiological Letters 123, 343-347 (1994)). After homologousrecombination by means of a “cross-over” event, the coding region of thegene involved is disrupted by the vector sequence and two incompletealleles are obtained, in which the 3′-or the 5′-ends respectively areeach missing. This method was used, for example, by Fitzpatrick et al.(Applied Microbiology and Biotechnology 42, 575-580 (1994)) to switchoff the recA gene in C. glutamicum.

[0082] With the method of gene replacement, a mutation such as e.g. adeletion, insertion or base replacement is produced in-vitro in the genebeing considered. The allele produced is again cloned in a vector whichdoes not replicate in C. glutamicum and this is then transferred bytransformation or conjugation into the desired host for C. glutamicum.After homologous recombination by means of a first, integration-causing“cross-over” event and an appropriate second, excision-causing“cross-over” event in the target gene or in the target sequence,incorporation of the mutation or the allele is achieved. This method wasused, for example, by Peters-Wendisch et al.(Microbiology 144, 915 - 927(1998)) to switch off the pyc gene in C. glutamicum by means of adeletion.

[0083] A deletion, insertion or base replacement can be incorporated inthe dep33 gene in this way.

[0084] In addition, it may be advantageous for the production of L-aminoacids, in addition to attenuating the dep33 gene in one or more enzymeson the relevant biosynthetic pathway, to enhance, in particularoverexpress, glycolysis, anaploretic processes, the citric acid cycle,the pentose-phosphate cycle, amino acid export and optionally regulatoryproteins.

[0085] The expression “enhancement” in this context describes theincrease in intracellular activity of one or more enzymes (proteins) ina microorganism which are coded by the corresponding DNA, for example byincreasing the copy number for the gene or genes, by using a strongpromoter or by using a gene or allele which codes for a correspondingenzyme (protein) with a high activity and optionally by combining thesemeasures.

[0086] Due to the measures for enhancement, in particularoverexpression, the activity or concentration of the correspondingprotein is generally increased by at least 10%, 25%, 50%, 75%, 100%,150%, 200%, 300%, 400% or 500%, with a maximum of up to 1000% or 2000%,with reference to the wild type protein or the activity or concentrationof the protein in the initially used microorganism.

[0087] Thus, to prepare L-amino acids, apart from attenuating the dep33gene, one or more of the genes chosen from the group consisting of

[0088] the dapA gene coding for dihydrodipicolinate synthase (EP-B 0 197335),

[0089] the gap gene coding for glyceraldehyde-3-phosphate dehydrogenase(Eikmanns (1992), Journal of Bacteriology 174:6076-6086),

[0090] the tpi gene coding for triosephosphate isomerase (Eikmanns(1992), Journal of Bacteriology 174:6076-6086),

[0091] the pgk gene coding for 3-phosphoglycerate kinase (Eikmanns(1992), Journal of Bacteriology 174:6076-6086),

[0092] the zwf gene coding for glucose-6-phosphate dehydrogenase(JP-A-09224661),

[0093] the pyc gene coding for pyruvate carboxylase (DE-A-198 31 609),

[0094] the mqo gene coding for malate quinone oxidoreduktase (Molenaaret al., European Journal of Biochemistry 254, 395-403 (1998)),

[0095] the lysC gene coding for a feed-back resistant aspartate kinase(Accession No.P26512; EP-B-0387527; EP-A-0699759; WO 00/63388),

[0096] the lysE gene coding for lysine export (DE-A-195 48 222),

[0097] the hom gene coding for homoserine dehydrogenase (EP-A 0131171),

[0098] the ilvA gene coding for threonine dehydratase (Mockel et al.,Journal of Bacteriology (1992) 8065-8072)) or the ilvA (Fbr) allelecoding for a “feed back resistant” threonine dehydratase (Möckel et al.,(1994) Molecular Microbiology 13: 833-842),

[0099] the ilvBN gene coding for acetohydroxyacid synthase (EP-B0356739),

[0100] the ilvD gene coding for dihydroxyacid dehydratase (Sahm andEggeling (1999) Applied and Environmental Microbiology 65: 1973-1979),

[0101] the zwal gene coding for Zwal protein (DE: 19959328.0, DSM 13115)

[0102] may be simultaneously enhanced, in particular overexpressed.

[0103] It may also be advantageous for the production of amino acids,apart from attenuating the dep33 gene, to simultaneously attenuate, inparticular to reduce the expression of, one or more genes chosen fromthe group consisting of

[0104] the pck gene coding for phosphoenolpyruvate carboxykinase (DE 19950 409.1, DSM 13047),

[0105] the pgi gene coding for glucose-6-phosphate isomerase (US09/396,478, DSM 12969),

[0106] the poxB gene coding for pyruvate oxidase (DE:1995 1975.7, DSM13114)

[0107] the zwa2 gene coding for Zwa2 protein (DE: 19959327.2, DSM13113).

[0108] Furthermore, it may be advantageous for the production of aminoacids, apart from attenuating the dep33 gene, to switch off undesiredside reactions (Nakayama: “Breeding of Amino Acid ProducingMicroorganisms”, in: Overproduction of Microbial Products, Krumphanzl,Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

[0109] Microorganisms prepared according to the invention are alsoprovided by the invention and may be cultivated continuously orbatchwise in a batch process or in a fed batch process or repeated fedbatch process for the purposes of producing L-amino acids. A review ofknown cultivation processes is given in the text book by Chmiel(Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik (GustavFischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas(Bioreaktoren und periphere Einrichtungen (Vieweg Verlag,Braunschweig/Wiesbaden, 1994)).

[0110] The culture medium to be used has to comply in a suitable mannerwith the requirements of the particular strain. Descriptions of culturemedia for different microorganisms are given in the manual “Manual ofMethods for General Bacteriology” by the American Society forBacteriology (Washington D.C., USA, 1981).

[0111] Sources of carbon which may be used are sugars and carbohydratessuch as e.g. glucose, saccharose, lactose, fructose, maltose, molasses,starch and cellulose, oils and fats such as, for example, soya oil,sunflower oil, peanut oil and coconut fat, fatty acids such as, forexample, palmitic acid, stearic acid and linoleic acid, alcohols suchas, for example, glycerine and ethanol and organic acids such as, forexample, acetic acid. These substances may be used individually or as amixture.

[0112] Sources of nitrogen which may be used are organicnitrogen-containing compounds such as peptones, yeast extract, meatextract, malt extract, maize steep liquor, soya bean meal and urea orinorganic compounds such as ammonium sulfate, ammonium chloride,ammonium phosphate, ammonium carbonate and ammonium nitrate. The sourcesof nitrogen may be used individually or as a mixture.

[0113] Sources of phosphorus which may be used are phosphoric acid,potassium dihydrogen phosphate or dipotassium hydrogen phosphate or thecorresponding sodium-containing salts. The culture medium must alsocontain salts of metals such as, for example, magnesium sulfate or ironsulfate, which are required for growth. Finally, essentialgrowth-promoting substances such as amino acids and vitamins may be usedin addition to the substances mentioned above. Suitable precursors maybe added to the culture medium in addition to these. The feedstuffsmentioned may be added to the culture in the form of a single batch orbe fed in a suitable manner during cultivation.

[0114] To regulate the pH of the culture, basic compounds such as sodiumhydroxide, potassium hydroxide, ammonia or ammoniacal liquor or acidcompounds such as phosphoric acid or sulfuric acid are used in anappropriate manner. To control the production of foam, antifoamingagents such as, for example, polyglycol esters of fatty acid may beused. To maintain the stability of plasmids, suitable selectively actingsubstances such as, for example, antibiotics, may be added to themedium. In order to maintain aerobic conditions, oxygen oroxygen-containing gas mixtures such as, for example, air, are passedinto the culture. The temperature of the culture is normally 20° C. to45° C. and is preferably 25° C. to 40° C. The culture procedure iscontinued until a maximum has been produced in the desired product. Thisobjective is normally achieved within 10 hours to 160 hours.

[0115] Methods for determining L-amino acids are known from the priorart. Analysis may be performed, for example, as described in Spackman etal. (Analytical Chemistry, 30, (1958), 1190) by anion exchangechromatography followed by ninhydrin derivation, or it may be performedby reversed phase HPLC as described in Lindroth et al. (AnalyticalChemistry (1979) 51: 1167-1174).

[0116] The process according to the invention is used for thefermentative preparation of amino acids.

[0117] The following microorganism was deposited on May 3, 2001 as apure culture at the German Collection of Microorganisms and CellCultures (DSMZ, Braunschweig, Germany) in accordance with the BudapestTreaty:

[0118]Escherichia coli top10/pCR2.1dep33int as DSM 14145.

[0119] The present invention is explained in more detail in thefollowing by using embodiment examples.

[0120] Isolation of plasmid DNA from Escherichia coli and all thetechniques for restriction, Klenow treatment and alkaline phosphatasetreatment were performed in the way described in Sambrook et al.(Molecular Cloning. A Laboratory Manual, 1989, Cold Spring HarbourLaboratory Press, Cold Spring Harbor, N.Y., USA). Methods for thetransformation of Escherichia coli are also described in this manual.

[0121] The composition of commonly used culture media such as LB mediumor TY medium may also be found in the manual by Sambrook et al.

EXAMPLE 1

[0122] Production of a Genomic Cosmid Gene Library from C. glutamicumATCC 13032

[0123] Chromosomal DNA from C. glutamicum ATCC 13032 was isolated asdescribed in Tauch et al., (1995, Plasmid 33:168-179), and partlycleaved with the restriction enzyme Sau3AI (Amersham Pharmacia,Freiburg, Germany, product description Sau3AI, Code no. 27-0913-02). TheDNA fragments were dephosphorylated with shrimp alkaline phosphatase(Roche Molecular Biochemicals, Mannheim, Germany, product descriptionSAP, Code no. 1758250). The DNA in the cosmid vector SuperCos1 (Wahl etal. (1987), Proceedings of the National Academy of Sciences, USA84:2160-2164), purchased from the Stratagene Co. (La Jolla, USA, productdescription SuperCos1 Cosmid Vektor Kit, Code no. 251301) was cleavedwith the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany,product description XbaI, Code no. 27-0948-02) and also dephosphorylatedwith shrimp alkaline phosphatase.

[0124] Then the cosmid DNA was cleaved with the restriction enzyme BamHI(Amersham Pharmacia, Freiburg, Germany, product description BamHI, Codeno. 27-0868-04). The cosmid DNA treated in this way was mixed with thetreated ATCC13032 DNA and the mixture was treated with T4 DNA ligase(Amersham Pharmacia, Freiburg, Germany, product descriptionT4-DNA-Ligase, Code no.27-0870-04). The ligation mixture was then packedinto phages with the aid of Gigapack II XL Packing Extracts (Stratagene,La Jolla, USA, product description Gigapack II XL Packing Extract, Codeno. 200217).

[0125] To infect E. coli strain NM554 (Raleigh et al. 1988, Nucleic AcidRes. 16:1563-1575), the cells were taken up in 10 mM MgSO₄ and mixedwith an aliquot of the phage suspension. Infection and titering of thecosmid library were performed as described in Sambrook et al. (1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), wherein thecells were plated out on LB agar (Lennox, 1955, Virology, 1:190)+100μg/ml ampicillin. After incubation overnight at 37° C., recombinantindividual clones were selected.

EXAMPLE 2

[0126] Isolating and Sequencing the Dep33 Gene

[0127] The cosmid DNA from an individual colony was isolated with theQiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany)in accordance with the manufacturer's information and partly cleavedwith the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg,Germany, product description Sau3AI, Product No. 27-0913-02). The DNAfragments were dephosphorylated with shrimp alkaline phosphatase (RocheMolecular Biochemicals, Mannheim, Germany, product description SAP,Product No. 1758250). After gel electrophoretic separation, isolation ofthe cosmid fragments in the size range 1500 to 2000 bp was performedwith QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden,Germany).

[0128] The DNA in sequencing vector pZero-1 purchased from theInvitrogen Co. (Groningen, Netherlands, product description ZeroBackground Cloning Kit, Product No. K2500-01) was cleaved with therestriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, productdescription BamHI, Product No. 27-0868-04). Ligation of the cosmidfragments in sequencing vector pZero-1 was performed as described inSambrook et al. (1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor), wherein the DNA mixture was incubated overnight with T4ligase (Pharmacia Biotech, Freiburg, Germany). This ligation mixture wasthen electroporated in E. coli strain DH5 MCR (Grant, 1990, Proceedingsof the National Academy of Sciences, U.S.A., 87:4645-4649) (Tauch et al.1994, FEMS Microbiol Letters, 123:343-7) and plated out on LB agar(Lennox, 1955, Virology, 1:190) with 50 μg/ml zeocin.

[0129] The plasmid preparation of recombinant clones was performed withBiorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany). Sequencingwas performed using the dideoxy chain termination method according toSanger et al. (1977, Proceedings of the National Academies of Sciences,U.S.A., 74:5463-5467) with modifications by Zimmermann et al. (1990,Nucleic Acids Research, 18:1067). The “RR dRhodamin Terminator CycleSequencing Kit” from PE Applied Biosystems(Product No. 403044,Weiterstadt, Germany) was used. Gel electrophoretic separation andanalysis of the sequencing reaction was performed in a “Rotiphorese NFAcrylamid/Bisacrylamid” Gel (29:1) (Product No. A124.1, Roth, Karlsruhe,Germany) using the “ABI Prism 377” sequencing instrument from PE AppliedBiosystems (Weiterstadt, Germany).

[0130] The crude sequencing data obtained were then processed using theStaden software package (1986, Nucleic Acids Research, 14:217-231)Version 97-0. The individual sequences of the pZerol derivatives wereassembled to give a cohesive Contig. Computer aided coding regionanalyses were drawn up with the program XNIP (Staden, 1986, NucleicAcids Research, 14:217-231). Further analyses were performed using the“BLAST search programs” (Altschul et al., 1997, Nucleic Acids Research,25:33893402) against the non-redundant database of the “National Centerfor Biotechnology Information” (NCBI, Bethesda, Md., USA).

[0131] The nucleotide sequence obtained is given in SEQ ID No. 1.Analysis of the nucleotide sequence produced an open reading frame of1635 bp, which was called the dep33 gene. The dep33 gene coded for apolypeptide of 544 amino acids.

EXAMPLE 3

[0132] Preparing an Integration Vector for Integration Mutagenesis ofthe Dep33 Gene

[0133] Chromosomal DNA was isolated from the strain ATCC 13032 using themethod described by Eikmanns et al. (Microbiology 140: 1817 -1828(1994)). Based on the sequence of the dep33 gene for C. glutamicum,known from example 2, the following oligonucleotides were chosen for thepolymerase chain reaction (see SEQ ID No. 3 and SEQ ID No. 4):

[0134] dep33-int1:

[0135] 5′ TGG ACT GAT GAT CCT CTC G 3′

[0136] dep33-int2:

[0137] 5′ AGG TAG GTC GGA AGG TAG C 3′

[0138] The primers shown were synthesized by MWG Biotech (Ebersberg,Germany) and the PCR reaction was performed using the standard PCRmethod described by Innis et al. (PCR protocols. A guide to methods andapplications, 1990, Academic Press) using Taq polymerase from BoehringerMannheim (Germany, product description Taq DNA Polymerase, Product No. 1146 165). With the aid of the polymerase chain reaction, the primersfacilitated amplification of a 531 bp sized internal fragment of thedep33 gene. The product amplified in this way was electrophoreticallytested in a 0.8% strength agarose gel.

[0139] The amplified DNA fragment was ligated with the TOPO TA cloningkit from the Invitrogen Corporation (Carlsbad, Calif., USA; cataloguenumber K4500-01) in vector pCR2.1-TOPO (Mead at al. (1991)Bio/Technology 9:657-663).

[0140] Then the E. coli strain TOP10 was electroporated with theligation mixture (Hanahan, In: DNA Cloning. A Practical approach. Vol.I, IRL-Press, Oxford, Washington D.C., USA, 1985). The selection ofplasmid-carrying cells was performed by plating out the transformationmixture on LB agar (Sambrook et al., Molecular cloning: a laboratorymanual. 2^(nd) Ed. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989) which had been supplemented with 50 mg/l kanamycin.Plasmid DNA was isolated from a transformant with the aid of the QIAprepSpin Miniprep kit from Qiagen and examined by restriction with therestriction enzyme EcoRI followed by agarose gel electrophoresis (0.8%).The plasmid was called pCR2.1dep33int and is shown in FIG. 1.

EXAMPLE 4

[0141] Integration Mutagenesis of the Dep33 Gene in the Strain DSM 5715

[0142] The vector called pCR2.1dep33int in example 3 was electroporatedinto Corynebacterium glutamicum DSM 5715 using the electroporationmethod described by Tauch et al.(FEMS Microbiological Letters,123:343-347 (1994)). The strain DSM 5715 is an AEC resistant lysineproducer. The vector pCR2.1dep33int cannot replicate itself in DSM5715and only remains in the cells when it has been integrated into thechromosome of DSM 5715. The selection of clones with pCR2.1dep33intintegrated into the chromosome was performed by plating out theelectroporation mixture on LB agar (Sambrook et al., Molecular cloning:a laboratory manual. 2^(nd) Ed. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.) which had been supplemented with 15 mg/lkanamycin.

[0143] To prove that integration had occurred, the dep33int fragment waslabeled with the Dig hybridization kit from Boehringer using the methoddescribed in “The DIG System Users Guide for Filter Hybridization” fromBoehringer Mannheim GmbH (Mannheim, Germany, 1993). Chromosomal DNA froma potential integrant was isolated using the method described byEikmanns et al. (Microbiology 140: 1817 -1828 (1994)) and each wasrestricted with the restriction enzymes BamHI, EcoRI and PstI. Thefragments produced were separated using agarose gel electrophoresis andwere hybridized at 68° C. with the Dig hybridization kit fromBoehringer. Plasmid pCR2.1dep33int mentioned in example 3 had insertedwithin the chromosomal dep33 gene in the chromosome of DSM5715. Thestrain was called DSM5715::pCR2.1dep33int.

EXAMPLE 5

[0144] Preparing Lysine

[0145] The C. glutamicum strain DSM5715::pCR2.1dep33int obtained inexample 4 was cultivated in a culture medium suitable for the productionof lysine and the lysine concentration in the culture supernatant liquidwas determined.

[0146] For this purpose, the strain was first incubated for 24 hours at33° C. on agar plates with the corresponding antibiotic (brain-heartagar with kanamycin (25 mg/l). Starting with these agar plate cultures,a preculture was inoculated (10 ml of medium in 100 ml conical flasks).The complete medium CgIII was used as the medium for the preculture.Medium Cg III NaCl 2.5 g/l Bacto peptone 10 g/l Bacto yeast extract 10g/l Glucose (autoclaved separately) 2% (w/v) The pH was adjusted to pH7.4

[0147] The pH was adjusted to pH 7.4

[0148] Kanamycin (25 mg/l) was added to this. The preculture wasincubated on the shaker at 33° C. for 16 hours at 240 rpm. A mainculture was inoculated with this preculture so that the initial OD (660nm) of the main culture was 0.1 OD. The medium MM was used for the mainculture. Medium MM CSL (Corn Steep Liquor) 5 g/l MOPS(Morpholinopropanesulfonic 20 g/l acid) Glucose (autoclaved separately)50 g/l Salts: (NH₄)₂SO₄ 25 g/l KH₂PO₄ 0.1 g/l MgSO₄ * 7 H₂O 1.0 g/lCaCl₂ * 2 H₂O 10 mg/l FeSO₄ * 7 H₂O 10 mg/l MnSO₄ * H₂O 5.0 mg/l Biotin(filtered sterile) 0.3 mg/l Thiamine * HCl (filtered sterile) 0.2 mg/lLeucine (filtered sterile) 0.1 g/l CaCO₃ 25 g/l

[0149] CSL, MOPS and the salt solution are adjusted to pH 7 withammoniacal liquor and autoclaved. Then the sterile substrate solutionand vitamin solution, and also the dry-autoclaved CaCO₃ are added.

[0150] Cultivation takes place in 10 ml volumes in a 100 ml conicalflask with baffles. Kanamycin (25 mg/l) was added.

[0151] Cultivation takes place at 33° C. and 80% atmospheric humidity.

[0152] After 72 hours, the OD was determined at a test wavelength of 660nm using the Biomek 1000 (Beckmann Instruments GmbH, Munich). The amountof lysine produced was determined with an amino acid analyzer fromEppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatographyand post-column derivation with ninhydrin detection.

[0153] Table 1 gives the results of the trial. TABLE 1 OD Lysine-HClStrain (660 nm) g/l DSM5715 8.5 12.64 DSM5715::pCR2.1dep33int 9.2 14.11

[0154] The abbreviations and names used are defined as follows: KmR:Kanamycin resistance gene BamHI: Restriction site of restriction enzymeBamHI EcoRI: Restriction site of restriction enzyme EcoRI PstI:Restriction site of restriction enzyme PstI dep33int: Internal fragmentof the dep33 gene ColE1: Replication origin of the plasmid ColE1

[0155] Obviously, numerous modifications and variations on the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

1 4 1 2100 DNA Corynebacterium glutamicum CDS (258)..(1889) 1 gaaaacgtccccgcagcagt ggcttccatg ggtgaagaag gcgcacaata cgcctcagca 60 atgtccgatttctccggtgc atccaacctc actccacacc ttgttgaatc acttccacaa 120 gcactccgtgaagcaattca actttcttac aacgacgccc tgacaccaat cttcttggcg 180 ctcaccccgatcgcagtagt cgccgcgatc ctcctctttt tcatccgtga agatcacctc 240 aaggaaacgcacgaata atg aca cac gaa act tcc gtc ccc gga cct gcc 290 Met Thr His GluThr Ser Val Pro Gly Pro Ala 1 5 10 gac gcg cag gtc gca gga gat acg aagctg cgc aaa ggc cgc gcg aag 338 Asp Ala Gln Val Ala Gly Asp Thr Lys LeuArg Lys Gly Arg Ala Lys 15 20 25 aag gaa aaa act cct tca tca atg acg cctgaa caa caa aag aaa gtc 386 Lys Glu Lys Thr Pro Ser Ser Met Thr Pro GluGln Gln Lys Lys Val 30 35 40 tgg tgg gtc ctc agc gcg ctg atg gtc gcc atgatg atg gcc tcc ctt 434 Trp Trp Val Leu Ser Ala Leu Met Val Ala Met MetMet Ala Ser Leu 45 50 55 gac cag atg att ttc ggc aca gcc ctg cca aca atcgtc ggt gaa ctc 482 Asp Gln Met Ile Phe Gly Thr Ala Leu Pro Thr Ile ValGly Glu Leu 60 65 70 75 ggc ggc gtt gac cac atg atg tgg gtc atc acc gcatac cta ctt gcc 530 Gly Gly Val Asp His Met Met Trp Val Ile Thr Ala TyrLeu Leu Ala 80 85 90 gaa acc atc atg ctg ccg atc tac gga aag ctc ggc gacctg gtt gga 578 Glu Thr Ile Met Leu Pro Ile Tyr Gly Lys Leu Gly Asp LeuVal Gly 95 100 105 cgt aaa ggt ctc ttc atc gga gcc ctc ggc atc ttc ctgatc ggc tcc 626 Arg Lys Gly Leu Phe Ile Gly Ala Leu Gly Ile Phe Leu IleGly Ser 110 115 120 gtc atc ggc ggg ctt gca gga aat atg acc tgg ttg atcgtc ggc cgt 674 Val Ile Gly Gly Leu Ala Gly Asn Met Thr Trp Leu Ile ValGly Arg 125 130 135 gcc gta cag ggc atc ggt ggc ggt gga ctg atg atc ctctcg cag gca 722 Ala Val Gln Gly Ile Gly Gly Gly Gly Leu Met Ile Leu SerGln Ala 140 145 150 155 atc atc gcg gac gtt gtt cca gca cgt gaa cgt ggccgc tac atg ggt 770 Ile Ile Ala Asp Val Val Pro Ala Arg Glu Arg Gly ArgTyr Met Gly 160 165 170 gtc atg ggt gga gtc ttc gga ctc tct gca gtt cttggc cca cta ctc 818 Val Met Gly Gly Val Phe Gly Leu Ser Ala Val Leu GlyPro Leu Leu 175 180 185 ggt ggc tgg ttc acc gaa gga cca ggc tgg cgc tgggca ttc tgg atg 866 Gly Gly Trp Phe Thr Glu Gly Pro Gly Trp Arg Trp AlaPhe Trp Met 190 195 200 aac atc cca ctg gga atc atc gcc atc ggt gtc gccatt tac ttc ctg 914 Asn Ile Pro Leu Gly Ile Ile Ala Ile Gly Val Ala IleTyr Phe Leu 205 210 215 gac att cca aag aag agc gtc aag ttc cgc tgg gattac ctg ggc act 962 Asp Ile Pro Lys Lys Ser Val Lys Phe Arg Trp Asp TyrLeu Gly Thr 220 225 230 235 ttc ttc atg atc gtt gcc gca acc agc ctg atcctg ttc acc acc tgg 1010 Phe Phe Met Ile Val Ala Ala Thr Ser Leu Ile LeuPhe Thr Thr Trp 240 245 250 ggt gga tcc cag tac gag tgg tct gat cca atcatc att gga ctg atc 1058 Gly Gly Ser Gln Tyr Glu Trp Ser Asp Pro Ile IleIle Gly Leu Ile 255 260 265 atc acc acc atc gtt gcc gct gca ctg ctg gttgtt gtg gaa ctg cgc 1106 Ile Thr Thr Ile Val Ala Ala Ala Leu Leu Val ValVal Glu Leu Arg 270 275 280 gca aaa gat cca ttg gtt cca atg tcc ttc ttccaa aac cgc aac ttc 1154 Ala Lys Asp Pro Leu Val Pro Met Ser Phe Phe GlnAsn Arg Asn Phe 285 290 295 acg ctc acc acc att gca ggc ctg atc ctg ggtatc gca atg ttc ggc 1202 Thr Leu Thr Thr Ile Ala Gly Leu Ile Leu Gly IleAla Met Phe Gly 300 305 310 315 atc atc ggc tac ctt ccg acc tac ctc cagatg gtc cac gga atc aac 1250 Ile Ile Gly Tyr Leu Pro Thr Tyr Leu Gln MetVal His Gly Ile Asn 320 325 330 gcc acc gaa gcc ggc tac atg ctg atc ccaatg atg gtc ggc atg atg 1298 Ala Thr Glu Ala Gly Tyr Met Leu Ile Pro MetMet Val Gly Met Met 335 340 345 ggt acc tcc atc tgg act ggt atc cgc atctcc aac aca gga aag tac 1346 Gly Thr Ser Ile Trp Thr Gly Ile Arg Ile SerAsn Thr Gly Lys Tyr 350 355 360 aaa ctc ttc cca cca atc ggc atg gtg gttacc ttc gtg gca ctg atc 1394 Lys Leu Phe Pro Pro Ile Gly Met Val Val ThrPhe Val Ala Leu Ile 365 370 375 ttc ttt gcc cga atg gaa gtg tcc acc accctg tgg cag atc gga atc 1442 Phe Phe Ala Arg Met Glu Val Ser Thr Thr LeuTrp Gln Ile Gly Ile 380 385 390 395 tac ctc ttc gtc ctc ggc gtc ggc ctgggt cta gcc atg cag gtt ctg 1490 Tyr Leu Phe Val Leu Gly Val Gly Leu GlyLeu Ala Met Gln Val Leu 400 405 410 gtc ctg atc gtt cag aac acc ctg ccaacc gcg gtg gtc gga tcc gca 1538 Val Leu Ile Val Gln Asn Thr Leu Pro ThrAla Val Val Gly Ser Ala 415 420 425 acc gct gtg aac aac ttc ttc cgt caaatc ggt tcc tca ctc gga tcc 1586 Thr Ala Val Asn Asn Phe Phe Arg Gln IleGly Ser Ser Leu Gly Ser 430 435 440 gcg ctg gtc ggt ggc atg ttc gtt ggcaac ttg gga acc ctc atg gaa 1634 Ala Leu Val Gly Gly Met Phe Val Gly AsnLeu Gly Thr Leu Met Glu 445 450 455 gaa aga atg cca gca gcc atg gca caactt tca cca gaa gaa caa gcc 1682 Glu Arg Met Pro Ala Ala Met Ala Gln LeuSer Pro Glu Glu Gln Ala 460 465 470 475 gcc atg gca gcc caa ggc gga ctggac tcc aac gaa ttg acg ccg gca 1730 Ala Met Ala Ala Gln Gly Gly Leu AspSer Asn Glu Leu Thr Pro Ala 480 485 490 atc gtc aat caa ttg cca acc gcgctc cac gat gcg ttc gcc ggt tcc 1778 Ile Val Asn Gln Leu Pro Thr Ala LeuHis Asp Ala Phe Ala Gly Ser 495 500 505 tac aac gac gca ctc atc cca gtgttc tac gtg atg atg cca ctg atc 1826 Tyr Asn Asp Ala Leu Ile Pro Val PheTyr Val Met Met Pro Leu Ile 510 515 520 ggc atc gcg ctg ctt ctc ttg ctgttt att aag caa gaa aaa cta cgc 1874 Gly Ile Ala Leu Leu Leu Leu Leu PheIle Lys Gln Glu Lys Leu Arg 525 530 535 gaa acc acc aca gac taaacacaaaacaaatgaga cctaccctcg ggtaggtctc 1929 Glu Thr Thr Thr Asp 540 atttgtttagggtcgcgtcg aaaagcaaaa agccttaatc aaagacaacc gtgcggttac 1989 cgtaaaccagcacccggtcc tccaagtgga aacgcaaacc gcgagccagc acctgcttct 2049 ccgcatcgcggcccaaacgc tgcatctcag tcggcgtatc cttatgcgtc a 2100 2 544 PRTCorynebacterium glutamicum 2 Met Thr His Glu Thr Ser Val Pro Gly Pro AlaAsp Ala Gln Val Ala 1 5 10 15 Gly Asp Thr Lys Leu Arg Lys Gly Arg AlaLys Lys Glu Lys Thr Pro 20 25 30 Ser Ser Met Thr Pro Glu Gln Gln Lys LysVal Trp Trp Val Leu Ser 35 40 45 Ala Leu Met Val Ala Met Met Met Ala SerLeu Asp Gln Met Ile Phe 50 55 60 Gly Thr Ala Leu Pro Thr Ile Val Gly GluLeu Gly Gly Val Asp His 65 70 75 80 Met Met Trp Val Ile Thr Ala Tyr LeuLeu Ala Glu Thr Ile Met Leu 85 90 95 Pro Ile Tyr Gly Lys Leu Gly Asp LeuVal Gly Arg Lys Gly Leu Phe 100 105 110 Ile Gly Ala Leu Gly Ile Phe LeuIle Gly Ser Val Ile Gly Gly Leu 115 120 125 Ala Gly Asn Met Thr Trp LeuIle Val Gly Arg Ala Val Gln Gly Ile 130 135 140 Gly Gly Gly Gly Leu MetIle Leu Ser Gln Ala Ile Ile Ala Asp Val 145 150 155 160 Val Pro Ala ArgGlu Arg Gly Arg Tyr Met Gly Val Met Gly Gly Val 165 170 175 Phe Gly LeuSer Ala Val Leu Gly Pro Leu Leu Gly Gly Trp Phe Thr 180 185 190 Glu GlyPro Gly Trp Arg Trp Ala Phe Trp Met Asn Ile Pro Leu Gly 195 200 205 IleIle Ala Ile Gly Val Ala Ile Tyr Phe Leu Asp Ile Pro Lys Lys 210 215 220Ser Val Lys Phe Arg Trp Asp Tyr Leu Gly Thr Phe Phe Met Ile Val 225 230235 240 Ala Ala Thr Ser Leu Ile Leu Phe Thr Thr Trp Gly Gly Ser Gln Tyr245 250 255 Glu Trp Ser Asp Pro Ile Ile Ile Gly Leu Ile Ile Thr Thr IleVal 260 265 270 Ala Ala Ala Leu Leu Val Val Val Glu Leu Arg Ala Lys AspPro Leu 275 280 285 Val Pro Met Ser Phe Phe Gln Asn Arg Asn Phe Thr LeuThr Thr Ile 290 295 300 Ala Gly Leu Ile Leu Gly Ile Ala Met Phe Gly IleIle Gly Tyr Leu 305 310 315 320 Pro Thr Tyr Leu Gln Met Val His Gly IleAsn Ala Thr Glu Ala Gly 325 330 335 Tyr Met Leu Ile Pro Met Met Val GlyMet Met Gly Thr Ser Ile Trp 340 345 350 Thr Gly Ile Arg Ile Ser Asn ThrGly Lys Tyr Lys Leu Phe Pro Pro 355 360 365 Ile Gly Met Val Val Thr PheVal Ala Leu Ile Phe Phe Ala Arg Met 370 375 380 Glu Val Ser Thr Thr LeuTrp Gln Ile Gly Ile Tyr Leu Phe Val Leu 385 390 395 400 Gly Val Gly LeuGly Leu Ala Met Gln Val Leu Val Leu Ile Val Gln 405 410 415 Asn Thr LeuPro Thr Ala Val Val Gly Ser Ala Thr Ala Val Asn Asn 420 425 430 Phe PheArg Gln Ile Gly Ser Ser Leu Gly Ser Ala Leu Val Gly Gly 435 440 445 MetPhe Val Gly Asn Leu Gly Thr Leu Met Glu Glu Arg Met Pro Ala 450 455 460Ala Met Ala Gln Leu Ser Pro Glu Glu Gln Ala Ala Met Ala Ala Gln 465 470475 480 Gly Gly Leu Asp Ser Asn Glu Leu Thr Pro Ala Ile Val Asn Gln Leu485 490 495 Pro Thr Ala Leu His Asp Ala Phe Ala Gly Ser Tyr Asn Asp AlaLeu 500 505 510 Ile Pro Val Phe Tyr Val Met Met Pro Leu Ile Gly Ile AlaLeu Leu 515 520 525 Leu Leu Leu Phe Ile Lys Gln Glu Lys Leu Arg Glu ThrThr Thr Asp 530 535 540 3 19 DNA Corynebacterium glutamicum 3 tggactgatgatcctctcg 19 4 19 DNA Corynebacterium glutamicum 4 aggtaggtcg gaaggtagc19

What is claimed is:
 1. An isolated polynucleotide, which encodes aprotein comprising the amino acid sequence of SEQ ID NO:2.
 2. Theisolated polynucleotide of claim 1, wherein said protein has Dep33efflux protein activity.
 3. A vector comprising the isolatedpolynucleotide of claim
 1. 4. A host cell comprising the isolatedpolynucleotide of claim
 1. 5. The host cell of claim 4, which is aCoryneform bacterium.
 6. The host cell of claim 4, wherein said hostcell is selected from the group consisting of Coryneform glutamicum,Corynebacterium acetoglutamicum, Corynebacterium acetoacidophilum,Corynebacterium melassecola, Corynebacterium thermoaminogenes,Brevibacterium flavum, Brevibacterium lactofermentum, and Brevibacteriumdivaricatum.
 7. A method for detecting a nucleic acid with at least 70%homology to nucleotide of claim 1, comprising contacting a nucleic acidsample with a probe or primer comprising at least 15 consecutivenucleotides of the nucleotide sequence of claim 1, or at least 15consecutive nucleotides of the complement thereof.
 8. A method forproducing a nucleic acid with at least 70% homology to nucleotide ofclaim 1, comprising contacting a nucleic acid sample with a primercomprising at least 15 consecutive nucleotides of the nucleotidesequence of claim 1, or at least 15 consecutive nucleotides of thecomplement thereof.
 9. A process for screening for polynucleotides,which encode a protein having Dep33 efflux activity comprising a)hybridizing the isolated polynucleotide of claim 1 to the polynucleotideto be screened; b) expressing the polynucleotide to produce a protein;and c) detecting the presence or absence of Dep33 efflux proteinactivity in said protein.
 10. A method for making a Dep33 effluxprotein, comprising culturing the host cell of claim 4 for a time andunder conditions suitable for expression of the Dep33 protein; andcollecting the Dep33 efflux protein.
 11. An isolated polynucleotide,which comprises SEQ ID NO:1.
 12. An isolated polynucleotide, which iscomplimentary to the polynucleotide of claim
 11. 13. An isolatedpolynucleotide, which is at least 70% identical to the polynucleotide ofclaim
 11. 14. An isolated polynucleotide, which is at least 80%identical to the polynucleotide of claim
 11. 15. An isolatedpolynucleotide, which is at least 90% identical to the polynucleotide ofclaim
 11. 16. An isolated polynucleotide, which comprises at least 15consecutive nucleotides of the polynucleotide of claim
 11. 17. Anisolated polynucleotide, which hybridizes under stringent conditions tothe polynucleotide of claim 11; wherein said stringent conditionscomprise washing in 5×SSC at a temperature from 50 to 68° C.
 18. Theisolated polynucleotide of claim 11, which encodes a protein havingDep33 efflux activity.
 19. A vector comprising the isolatedpolynucleotide of claim
 11. 20. A host cell comprising the isolatedpolynucleotide of claim
 11. 21. The host cell of claim 20, which is aCoryneform bacterium.
 22. The host cell of claim 20, wherein said hostcell is selected from the group consisting of Coryneform glutamicum,Corynebacterium acetoglutamicum, Corynebacterium acetoacidophilum,Corynebacterium melassecola, Corynebacterium thermoaminogenes,Brevibacterium flavum, Brevibacterium lactofermentum, and Brevibacteriumdivaricatum.
 23. A process for screening for polynucleotides, whichencode a protein having Dep33 efflux activity comprising a) hybridizingthe isolated polynucleotide of claim 11 to the polynucleotide to bescreened; b) expressing the polynucleotide to produce a protein; and c)detecting the presence or absence of Dep33 efflux protein activity insaid protein.
 24. A method for detecting a nucleic acid with at least70% homology to nucleotide of claim 11, comprising contacting a nucleicacid sample with a probe or primer comprising at least 15 consecutivenucleotides of the nucleotide sequence of claim 11, or at least 15consecutive nucleotides of the complement thereof.
 25. A method forproducing a nucleic acid with at least 70% homology to nucleotide ofclaim 11, comprising contacting a nucleic acid sample with a primercomprising at least 15 consecutive nucleotides of the nucleotidesequence of claim 11, or at least 15 consecutive nucleotides of thecomplement thereof.
 26. A method for making Dep33 efflux protein,comprising a) culturing the host cell of claim 20 for a time and underconditions suitable for expression of the Dep33 efflux protein; and b)collecting the Dep33 efflux protein.
 27. A Coryneform bacterium, whichcomprises an attenuated dep33 gene.
 28. The Coryneform bacterium ofclaim 27, wherein said dep33 gene comprises the polynucleotide sequenceof SEQ ID NO:1.
 29. Escherichia coli DSM
 14145. 30. A process forproducing L-amino acids comprising culturing a bacterial cell in amedium suitable for producing L-amino acids, wherein said bacterial cellcomprises an attenuated dep33 gene.
 31. The process of claim 30, whereinsaid bacterial cell is a Coryneform bacterium or Brevibacterium.
 32. Theprocess of claim 31, wherein said bacterial cell is selected from thegroup consisting of Coryneform glutamicum, Corynebacteriumacetoglutamicum, Corynebacterium acetoacidophilum, Corynebacteriummelassecola, Corynebacterium thermoaminogenes, Brevibacterium flavum,Brevibacterium lactofermentum, and Brevibacterium divaricatum.
 33. Theprocess of claim 30, wherein said dep33 gene comprises thepolynucleotide sequence of SEQ ID NO:1.
 34. The process of claim 30,wherein said L-amino acid is L-lysine.
 35. The process of claim 30,wherein said bacteria further comprises at least one gene whoseexpression is enhanced, wherein said gene is selected from the groupconsisting of dapA, gap, tp1, pgk, zwf, pyc, mqu, lysC, lysE, hom, ilvA,ilvBN, ilvD and zwa
 1. 36. The process of claim 30, wherein saidbacteria further comprises at least one gene whose expression isattenuated, wherein said gene is selected from the group consisting ofpck, pgi, poxB, and zwa2.
 37. An isolated polypeptide comprising theamino acid sequence of SEQ ID NO:2.